Tied down: tethering redox proteins to the outer membrane in Neisseria and other genera

2011 ◽  
Vol 39 (6) ◽  
pp. 1895-1899 ◽  
Author(s):  
Xi Li ◽  
Steven Parker ◽  
Manu Deeudom ◽  
James W. Moir
Keyword(s):  
Outer Membrane ◽  
Host Response ◽  
Cytoplasmic Membrane ◽  
Gram Negative Bacteria ◽  
Globular Domain ◽  
Redox Proteins ◽  
Organizational Arrangement ◽  
Functional Consequences ◽  
Repeated Structure ◽  
Respiratory Chains

Typically, the redox proteins of respiratory chains in Gram-negative bacteria are localized in the cytoplasmic membrane or in the periplasm. An alternative arrangement appears to be widespread within the betaproteobacterial genus Neisseria, wherein several redox proteins are covalently associated with the outer membrane. In the present paper, we discuss the structural properties of these outer membrane redox proteins and the functional consequences of this attachment. Several tethered outer membrane redox proteins of Neisseria contain a weakly conserved repeated structure between the covalent tether and the redox protein globular domain that should enable the redox cofactor-containing domain to extend from the outer membrane, across the periplasm and towards the inner membrane. It is argued that the constraints imposed on the movement and orientation of the globular domains by these tethers favours the formation of electron-transfer complexes for entropic reasons. The attachment to the outer membrane may also affect the exposure of the host to redox proteins with a moonlighting function in the host–microbe interaction, thus affecting the host response to Neisseria infection. We identify putative outer membrane redox proteins from a number of other bacterial genera outside Neisseria, and suggest that this organizational arrangement may be more common than previously recognized.

scholarly journals A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

2019 ◽  
Vol 116 (43) ◽  
pp. 21748-21757 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Angela M. Mitchell ◽  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Jessica Sheng ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Small Molecule ◽  
Cytoplasmic Membrane ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bam Complex ◽  
Induced Aggregation

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

scholarly journals Depolarization, Bacterial Membrane Composition, and the Antimicrobial Action of Ceragenins

2010 ◽  
Vol 54 (9) ◽  
pp. 3708-3713 ◽  
Author(s):  
Raquel F. Epand ◽  
Jake E. Pollard ◽  
Jonathan O. Wright ◽  
Paul B. Savage ◽  
Richard M. Epand
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Bacterial Species ◽  
Membrane Depolarization ◽  
Membrane Lipid ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Cytoplasmic Membranes

ABSTRACT Ceragenins are cholic acid-derived antimicrobial agents that mimic the activity of endogenous antimicrobial peptides. Ceragenins target bacterial membranes, yet the consequences of these interactions have not been fully elucidated. The role of the outer membrane in allowing access of the ceragenins to the cytoplasmic membrane of Gram-negative bacteria was studied using the ML-35p mutant strain of Escherichia coli that has been engineered to allow independent monitoring of small-molecule flux across the inner and outer membranes. The ceragenins CSA-8, CSA-13, and CSA-54 permeabilize the outer membrane of this bacterium, suggesting that the outer membrane does not play a major role in preventing the access of these agents to the cytoplasmic membrane. However, only the most potent of these ceragenins, CSA-13, was able to permeabilize the inner membrane. Interestingly, neither CSA-8 nor CSA-54 caused inner membrane permeabilization over a 30-min period, even at concentrations well above those required for bacterial toxicity. To further assess the role of membrane interactions, we measured membrane depolarization in Gram-positive bacteria with different membrane lipid compositions, as well as in Gram-negative bacteria. We found greatly increased membrane depolarization at the minimal bactericidal concentration of the ceragenins for bacterial species containing a high concentration of phosphatidylethanolamine or uncharged lipids in their cytoplasmic membranes. Although membrane lipid composition affected bactericidal efficiency, membrane depolarization was sufficient to cause lethality, providing that agents could access the cytoplasmic membrane. Consequently, we propose that in targeting bacterial cytoplasmic membranes, focus be placed on membrane depolarization as an indicator of potency.

On conducting electron traffic across the periplasm

2012 ◽  
Vol 40 (6) ◽  
pp. 1178-1180 ◽  
Author(s):  
Jeffrey A. Gralnick
Keyword(s):  
Physical Properties ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Respiratory Pathway ◽  
Redox Proteins ◽  
The Core ◽  
Core Components ◽  
Reducing Bacteria ◽  
Metal Reducing Bacteria

Dissimilatory metal-reducing bacteria are able to conduct electrons from their cytoplasmic membrane across the periplasm and the outer membrane to redox proteins located on the surface of their cells. The Mtr respiratory pathway in Shewanella is the best-understood metal-reducing pathway to date. The core components of this pathway are well agreed upon, but are they sufficient? Could there be other components that we have yet to uncover? The present paper specifically considers the periplasm, its physical properties and organization. Two models are presented to explain how electrons could be conducted across this compartment in Shewanella.

scholarly journals Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

2017 ◽  
Vol 199 (10) ◽  
Author(s):  
Michael G. Gresock ◽  
Kathleen Postle
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Current Model ◽  
Membrane Transporters ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Interaction Sites ◽  
Outer Membrane Transporters

ABSTRACT In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ∼158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligand-sensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivo. IMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as FepA. Only one contact between TonB and outer membrane transporters has been identified to date: the TonB box at the transporter amino terminus. The TonB box has low information content, raising the question of how TonB can discriminate among multiple different TonB-dependent transporters present in the bacterium if it is the only means of contact. Here we identified several additional sites through which FepA contacts TonB in vivo, including two neighboring residues that may explain how FepA signals to TonB that ligand has bound.

scholarly journals The Outer Membrane of Gram-Negative Bacteria Inhibits Antibacterial Activity of Brochocin-C

1999 ◽  
Vol 65 (10) ◽  
pp. 4329-4333 ◽  
Author(s):  
Yan Gao ◽  
Marco J. van Belkum ◽  
Michael E. Stiles
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Inhibitory Effect ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Serovar Typhimurium ◽  
Activity Spectrum ◽  
The Difference ◽  
Brochothrix Campestris ◽  
Hydrolysis Of

ABSTRACT Brochocin-C is a two-peptide bacteriocin produced byBrochothrix campestris ATCC 43754 that has a broad activity spectrum comparable to that of nisin. Brochocin-C has an inhibitory effect on EDTA-treated gram-negative bacteria, Salmonella enterica serovar Typhimurium lipopolysaccharide mutants, and spheroplasts of Typhimurium strains LT2 and SL3600. Brochocin-C treatment of cells and spheroplasts of strains of LT2 and SL3600 resulted in hydrolysis of ATP. The outer membrane of gram-negative bacteria protects the cytoplasmic membrane from the action of brochocin-C. It appears that brochocin-C is similar to nisin and possibly does not require a membrane receptor for its function; however, the difference in effect of the two bacteriocins on intracellular ATP indicates that they cause different pore sizes in the cytoplasmic membrane.

scholarly journals Method for Estimation of Low Outer Membrane Permeability to β-Lactam Antibiotics

2002 ◽  
Vol 46 (9) ◽  
pp. 2901-2907 ◽  
Author(s):  
Bernard Lakaye ◽  
Alain Dubus ◽  
Bernard Joris ◽  
Jean-Marie Frère
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Quantitative Estimation ◽  
Enterobacter Aerogenes ◽  
Gram Negative Bacteria ◽  
Penicillin Binding Protein ◽  
Highly Sensitive ◽  
Outer Membrane Permeability ◽  
Rate Limiting

ABSTRACT The outer membrane of gram-negative bacteria plays a major role in β-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with β-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated β-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of β-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of β-lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10−7 cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P = 3.2 × 10−9 cm/s). Using these values, the MICs for a β-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method.

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